O-linked protein glycosylation involves an initiation stage in which a family of N-acetylgalactosaminyltransferases catalyzes the addition of N-acetylgalactosamine to serine or threonine residues (1). Further assembly of O-glycan chains involves several sucessive or alternative biosynthetic reactions: i) formation of simple mucin-type core 1 structures by UDP-Gal: GalNAcα-R β1,3Gal-transferase activity; ii) conversion of core 1 to complex-type core 2 structures by UDP-GlcNAc: Galβ1-3GalNAcα-R β1,6GlcNAc-transferase activities; iii) direct formation of complex mucin-type core 3 by UDP-GlcNAc: GlcNAcα β1,3GlcNAc-transferase activities; and iv) conversion of core 3 to core 4 by UDP-GlcNAc: GlcNAcβ1-3GalNAcα-R β1,6GlcNAc-transferase activity. The formation of 1,6GlcNAc branches (reactions ii and iv) may be considered a key controlling event of O-linked protein glycosylation leading to structures produced upon differentiation and malignant transformation (2-6). For example, increased formation of GlcNAcβ1-6GalNAc branching in O-glycans has been demonstrated during T-cell activation, during the development of leukemia, and for immunodeficiencies like Wiskott-Aldrich syndrome and AIDS (7; 8). Core 2 branching may play a role in tumor progression and metastasis (9). In contrast, many carcinomas show changes from complex O-glycans found in normal cell types to immaturely processed simple mucin-type O-glycans such as T (Thomsen-Friedenreich antigen; Gal 1-3GalNAc 1-R), Tn (GalNAc 1-R), and sialosyl-Tn (NeuAc 2-6GalNAc 1-R) (10). The molecular basis for this has been extensively studied in breast cancer, where it was shown that specific downregulation of core 2 β6GlcNAc-transferase was responsible for the observed lack of complex type O-glycans on the mucin MUCl (6). O-glycan core assembly may therefore be controlled by inverse changes in the expression level of Core-β1,6-N-acetylglucosaminyltransferases and the sialyltransferases forming sialyl-T and sialyl-Tn.
Interestingly, the metastatic potential of tumors has been correlated with increased expression of core 2 β6GlcNAc-transferase activity (5). The increase in core 2 β6GlcNAc-transferase activity was associated with increased levels of poly N-acetyllactosamine chains carrying sialyl-Lex, which may contribute to tumor metastasis by altering selectin mediated adhesion (4; 11). The control of O-glycan core assembly is regulated by the expression of key enzyme activities outlined in FIG. 1; however, epigenetic factors including posttranslational modification, topology, or competition for substrates may also play a role in this process (11).
The in vitro biosynthesis of a subset of complex O-glycopeptide structures is presently hampered by lack of availability of the enzymes adding N-acetylglucosamine in a β1-3 linkage to GalNAcα1-O-Ser/Thr to form core 3 as well as the enzyme catalyzing the successive addition of β1-6 N-acetylglucosamine branches to form core 4. This structure is required for the enzymes responsible for further build-up of core 4 based complex type O-glycans (FIG. 1). Most other enzymes required for elongation of branched O-glycans are available, and the core 2/4 enzyme described herein now makes the synthesis of core 4 based structures possible.
Access to the gene encoding C2/4GnT would allow production of a glycosyltransferase for use in formation of core 2 or core 4—based O-glycan modifications on oligosacccharides, glycoproteins and glycosphingolipids. This enzyme could be used, for example in pharmaceutical or other commercial applications that require synthetic addition of core 2 or core 4 based O-glycans to these or other substrates, in order to produce appropriately glycosylated glycoconjugates having particular enzymatic, immunogenic, or other biological and/or physical properties.
Consequently, there exists a need in the art for UDP-N-Acetylglucosamine: Galactose-β1,3-N-Acetylgalactosamine-α-R/N-Acetylglucosamine-β1,3-N-Acetyl-galactosamine-α-R (GlcNAc to GalNAc) β1-6 N-Acetylglucosaminyltransferase and the primary structure of the gene encoding these enzyme. The present invention meets this need, and further presents other related advantages.